NASA Technical Reports Server (NTRS) 20010003440: Role of Integrin in Mechanical Loading of Osteoblasts

Final Technical Report

NASA Grant NAG5-6374

Principal Investigator: Ruth Globus, Ph.D.

Mechanical forces generated by gravity, weightbearing, and muscle contraction play a
key role in the genesis and maintenance of skeletal structure. Increased mechanical
loading caused by exercise stimulates osteoblasts resulting in increased bone formation
and accretion of skeletal mass. Conversely, astronauts exposed to prolonged space flight
suffer from site-selective osteopenia, which has been shown in growing rats to result
from reduced bone formation by osteoblasts. The reduction in bone formation appears to
be caused by defects at several stages of osteoblast differentiation, including
proliferation, matrix production, and mineralization. The molecular mechanisms that
mediate changes in osteoblast activity in response to altered patterns of skeletal loading
are not known, and a better understanding of these processes may be essential for
developing effective treatment strategies to prevent disuse osteoporosis.

The long-term goal of our collaborative research program is to understand how the
extracellular matrix (ECM) and cell adhesion proteins, integrins, interact to mediate the
response of osteoblasts and their progenitors to mechanical loading. We suggest that
integrin/ECM interactions are crucial for basic cellular processes, including
differentiation and survival, as well as to participate in detecting and mediating cellular
responses to mechanical stimuli.

Major Findings

As a first approach to determine the role of integrin/extracellular matrix (ECM)
interactions in bone formation, we analyzed the repertoire of integrins expressed in bone
and the influence of integrin/ECM perturbing factors on osteoblast differentiation in
vitro. Osteoblasts in fetal rat calvaria, and cultures derived from this tissue, express a
large number of different integrin receptors for fibronectin, collagen, and other ECM
ligands, including aipi, a2pi, a3pi, a5pi, a8pi, aVp3 andaVp5.

Integrin complexes detected on rat calvarial cells in vivo and in vitro and their ECM ligands
a 1 P 1 collagen, laminin

a2pi collagen, laminin

a3pi laminin, fibronectin, thrombospondin, collagen
a5pi fibronectin

a8pi fibronectin, vitronectin, tenascin

aVp3 vitronectin, fibronectin, fibrinogen, osteopontin, bone sialoprotein
aVp5 vitronectin, fibronectin

Primary cultures of fetal rat osteoblasts progressively differentiate in culture to
form mineralized nodules . To dissect the role of specific ECM proteins, we added
function-perturbing antibodies and fragments of ECM ligands to primary osteoblasts at
different stages of differentiation. We found that the addition to immature osteoblasts of
antibodies to a3pi, a5pi, and a8pi (but not aVp3, aVp5, a4pl), as well as soluble
ligands (fibronectin, laminin), ligand antibodies, and RGD peptides (which interfere with
integrin/ECM binding), inhibits both the expression of genes characteristic of the
osteoblast phenotype and the formation of mineralized nodules .

1

To determine if FN also plays an important role in the function of mature
osteoblasts as well as during differentiation, osteoblasts that had already formed
mineralized nodules in vitro were treated with FN antagonists FN antibodies (FNAb)
caused >95% of the cells in mature cultures to display characteristic features of apoptosis
within 24 h (nuclear condensation, apoptotic body formation, DNA laddering). Cells
appeared to acquire sensitivity to FNAb-induced apoptosis as a consequence of
differentiation, since FNAb failed to kill immature cells and the first cells killed by FNAb
were those associated with mature nodules. Intact plasma FN, as well as fragments
corresponding to the amino-terminal, cell-binding, and carboxy-terminal domains of FN,
independently induced apoptosis of mature, but not immature, osteoblasts. Thus FN
appears to function in the mature, but not the immature, ECM to sustain osteoblast
survival. Finally, transforming growth factor-131 partially protected cells from the
apoptotic effects of FNAb, indicating that TGF-6 may function in concert with FN, to
promote osteoblast survival in vivo. We conclude that FN functions to promote survival
of osteoblasts once they have matured, and that this may contribute to the regulation of
bone formation. Interestingly, the same anti-integrin antibodies and RGD peptides that
inhibit differentiation of immature cells fail to induce apoptosis of mature osteoblasts.
Thus, mature osteoblasts appear to rely on multiple interactions with the ECM to ensure
their survival in vitro.

Summary: Influence of Integrin/ECM Antagonists on Osteoblast Differentiation and Survival (see
references below)

Additive:

Differentiation of
immature Ob

Survival of
mature Ob

FN Antagonist (Ab, FN)

4

4

LN Antagonist (Ab, LN)

4

4

RGD Peptide

4 "

cc3pi Ab

4

cxSpi Ab

4

cx8pi Ab

4

aVp3 Ab

?

aVp5 Ab

?

To evaluate the effects of mechanical strain applied in vitro, studies were
performed using a commercially-available apparatus (Flexercell strain unit) using
Flexcell flexible dishes for both control and mechanically active conditions to generate
both compressive and tensile strains on primary osteoblasts. The expression of specific
ECM, integrin and cytoskeletal components in response to mechanical forces (4%
maximum deformation, 0.5 cycles/sec) was analyzed using immunocytochemical
techniques. We found that consistent changes in the pattern of expression of fibronectin,
a5 integrin, and actin are not evident after 2hr, 2d or 4d of strain relative to stationary
controls. Since the mechanical strain provided by the Flexcell system is non-uniform and
provides strain levels that greatly exceeded physiologic levels for bone we developed a
novel strain unit that can: 1) apply a predominantly uniaxial and uniform load to the
culture substrate at strains thought to be physiological for bone in vivo 2) dynamically
load cells with cyclic tensile and compressive mechanical deformation 3) permit real-
time microscopic observations by confocal imaging, without interrupting the loading

2

cycle. This loading unit is useful for evaluating integrin-ECM interactions that mediate
cellular responses to mechanical strain.

In conclusion, we have made substantial progress in elucidating those integrin/ECM
interactions that are needed for osteoblast function and have developed a useful loading
system to further explore the molecular basis of mechano-sensitivity of osteoblasts.

Peer-Reviewed Journal Articles

Moursi, A., Zimmerman, D., Lull , J., Damsky, C., and Globus, R.K. (1996) Fibronectin
regulates gene expression and progressive differentiation of calvarial osteoblasts. J. Cell
Sci., 109, 1369-1380.

Moursi AM, Globus RK, Lull J, Damsky CH. (1997) The a5pl integrin fibronectin
receptor regulates osteoblast differentiation. J Cell Sci 1 10:2187-2196.

Globus RK, Doty SB, Lull JC, Holmuhamedov E, Humphries MJ, Damsky CH (1998)
Fibronectin is a survival factor for differentiated osteoblasts. J Cell Sci 111 : 1385-1393.

Hammond TG, Lewis FC, Goodwin TJ, Linnehan RM, Wolf DA, Hire KP, Campbell
WC, Benes E, O'Reilly KC, Globus RK, Kaysen JH. (1999) Gene Expression in Space.
Nature Medicine (letter) 5;359

Proceedings/Reviews

Globus, R.K., Moursi, A., Zimmerman, D., Lull, J., Damsky, C. (1995) Integrin-
extracellular matrix interactions in connective tissue remodeling and osteoblast
differentation. ASGSB Bull., 8, 19-28.

Damsky CH, Moursi A, Zhou Y, Fisher SJ, Globus RK. (1997) The solid state
environment orchestrates embryonic development and tissue remodeling. Kidney Int
51:1427-1433.

Globus RK, Moursi A, Lopez V, Damsky CH. Role of fibronectin and adhesion receptors
in osteoblast differentiation and function. 1998. Biological Mechanisms of Tooth
Eruption, Resorption and Replacement by Implants. Ed Z. Davidovitch and J. Mah, pages
353-360. 1998. Harvard Society for the Advancement of Orthodontics, Boston, MA.

Morey-Holton ER, Globus RK. (1998) Skeletal changes during hindlimb unloading of
growing rats: A review. Bone 22:83S-88S

Abstracts/SpeakingEngagements/T echnical Memoranda

Gordon Conference on Gravitational Effects on Living Systems, New London, New
Hampshire (1994) Travel Award

3

Moursi A., Globus R., Lull J, Zimmerman D, Damsky C (1994). Expression and function
of integrins during osteogenesis. Mol Biol Cell 5:1835.

Moursi A., Globus R., Lull J, Zimmerman D, Damsky C (1994). Regulated integrin
expression and function during osteoblast differentiation. J Bone Min Res 8:175.

Globus, RK, Moursi, A, Lull JC, Zimmerman D, Doty SB, Damsky CH (1995)
Fibronectin regulates gene expression and progressive differentiation of osteoblasts. J
Bone Min Res 10:S219.

Malouvier A, Globus RK, Doty S, Lull J, Morey-Holton E (1995) Gravity regulates
glucose and lactate metabolism in cultured osteoblasts. ASGSB Bull 9:28

Doty SB, Boskey A, Binderman I, Globus RK, Holton EM (1995) The effect of
spaceflight on bone and cartilage cell differentiation. 5th International Conference on
Mineralized Tissues. Oct. 22, 1995.

Globus RK, Holmuhamedov E, Moursi A, Lull J, Damsky C. (1996) Fibronectin
suppresses apoptosis of mature calvarial osteoblasts. Mol Biol Cell, 5 (Suppl):564a

Moursi AM, Globus RK, Lull J, Damsky C (1996) Integrin receptors for fibronectin regulate
osteogenesis. J Bone Min Res, 1 l(Suppl.l):S137

Searby, NE. Morey-Holton, R. Globus, E. Holmuhamedov. Adaptation of bone to
mechanical stimulation: development and characterization of a unique osteoblast loading
system. NASA Technical Memorandum 1 10445, Director’s Discretionary Fund Report
for Fiscal Year 1996, Ames Research Center, March 1997.

Globus RK. Role of fibronectin and its adhesion receptors in the maturation and function
of cultured osteoblasts. Second International Conference on Biological Mechanisms of
Tooth Eruption, Resorption and Replacement by Implants. Travel Award, Oct. 1997.
Madrid Spain.

Globus RK, Doty SB, Damsky CH (1997) Disruption of fibronectin interactions causes
differentiation-dependent apoptosis of cultured osteoblasts. J Bone Min Res. 12:176.

Zimmerman D, Globus RK, Damsky CH (1997) In vivo and in vitro models of altered
integrin function in osteoblasts: effects on osteoblast maturation and bone remodelling. J
Bone Min Res. 12:P211.

Moursi AM, Najafi M, Globus RK, Damsky C (1997) Integrin-fibronectin interactions
are critical for osteogenesis and osteoblast differentiation. J Dental Res 76:296.

Globus RK. (1997) Fibronectin and osteoblast survival. Gordon Conference on
Fibronectin, Integrins, and Related Molecules. Invited speaker.

4

Globus RK, Holmuhamedov EL, Morey-Holton E and Moursi AM. (1998) Laminin as an
autocrine factor for the differentiation and survival of osteoblasts. American Society for
Bone and Mineral Research. Vol. 14 Supplement.

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NASA Grant NAG5-6374
Principal Investigator: Ruth Globus, Ph.D.

Abstract:

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gey role in the genesis altered patterns of stale.*

that mediate changes in Meoblas t *c0 ty P my be essence for

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the molecular basis of mechano-sensitivity of osteoblasts.